Liver type 1 innate lymphoid cells lacking IL-7 receptor are a native killer cell subset fostered by parenchymal niches

Group 1 innate lymphoid cells (G1-ILCs), including circulating natural killer (NK) cells and tissue-resident type 1 ILCs (ILC1s), are innate immune sentinels critical for responses against infection and cancer. In contrast to relatively uniform NK cells through the body, diverse ILC1 subsets have been characterized across and within tissues in mice, but their developmental and functional heterogeneity remain unsolved. Here, using multimodal in vivo approaches including fate-mapping and targeting of the interleukin 15 (IL-15)-producing microenvironment, we demonstrate that liver parenchymal niches support the development of a cytotoxic ILC1 subset lacking IL-7 receptor (7 R− ILC1s). During ontogeny, fetal liver (FL) G1-ILCs arise perivascularly and then differentiate into 7 R− ILC1s within sinusoids. Hepatocyte-derived IL-15 supports parenchymal development of FL G1-ILCs to maintain adult pool of 7 R− ILC1s. IL-7R+ (7R+) ILC1s in the liver, candidate precursors for 7 R− ILC1s, are not essential for 7 R− ILC1 development in physiological conditions. Functionally, 7 R− ILC1s exhibit killing activity at steady state through granzyme B expression, which is underpinned by constitutive mTOR activity, unlike NK cells with exogenous stimulation-dependent cytotoxicity. Our study reveals the unique ontogeny and functions of liver-specific ILC1s, providing a detailed interpretation of ILC1 heterogeneity.

Functional differences between NK cells and ILC1s have also been recognized, though some confusion remains, particularly in cytotoxicity. In mice, NK cells are traditionally considered as more cytotoxic than ILC1s (Vivier et al., 2018), although this view has been questioned recently (Dadi et al., 2016;Kansler et al., 2022;Krabbendam et al., 2021;Nixon et al., 2022;Yomogida et al., 2021). Indeed, murine NK cells show low expression of cytotoxic molecules and only minimal cytotoxicity in their steady state (Fehniger et al., 2007). The cytotoxicity of NK cells requires the mTOR-dependent metabolic reprogramming mediated by cytokine signaling such as IL-15 or by NK receptor engagement (Marçais et al., 2014;Nandagopal et al., 2014), but whether such cytotoxic machinery also exists in ILC1s is unclear. By contrast, ILC1s can immediately respond and produce IFN-γ during liver injury (Nabekura et al., 2020) and virus infection (Weizman et al., 2017), highlighting the unique roles of ILC1s in the ignition of type 1 immunity in tissues. Thus, examining the development, function, and heterogeneity of ILC1s could lead to further understanding of local immune regulation and novel therapeutic strategies.
Additionally, environmental factors regulating ILC1 development are poorly understood. Accumulating evidence has shown that development and maintenance of ILCs are strictly associated with their resident tissue microenvironment, called niche (Ikuta et al., 2021;McFarland and Colonna, 2020;Murphy et al., 2022). G1-ILC homeostasis heavily depends on interleukin 15 (IL-15), that is transpresented from hematopoietic and stromal cells as an IL-15/IL-15Rα complex to locally promote the development, survival, and proliferation of memory CD8 T cells, NKT cells, and G1-ILCs in various tissues (Ikuta et al., 2021;Klose et al., 2014;Lodolce et al., 1998). However, how tissue environment regulates ILC1 homeostasis and whether specific niches control the formation of ILC1 heterogeneity are yet to be characterized.
Based on fate-mapping, transfer studies, and targeting of the IL-15-producing microenvironment, we have addressed the developmental processes of heterogenous ILC1 subsets. Adult liver (AL) 7R + ILC1s are not converted to 7 R − ILC1s in vivo and RORα deficiency results in selective reduction of 7R + ILC1s, suggesting that 7R + ILC1s are not necessary for the development of 7 R − ILC1s. FL G1-ILCs originate from perivascular sites of the liver and then infiltrate into sinusoids to give rise to AL 7 R − ILC1s. Hepatocyte-derived IL-15 supports FL G1-ILCs development in parenchyma, thereby maintaining mature 7 R − ILC1s in sinusoids. Functionally, 7 R − ILC1s exert inflammation-independent cytotoxicity through granzyme B expression, which is underpinned by their tonic mTOR activity. Our findings reveal that 7 R − ILC1s represent an ILC subset with unique developmental processes and unconventional native cytotoxicity distinct from NK cells and 7R + ILC1s.
Given the ILC1-like transcriptional programs and T-bet + Eomes − phenotype of FL G1-ILCs and AL 7 R − ILC1s ( Figure 1G), they were considered as ILC1s but not NK cells. To precisely verify their lineage, we performed fate-mapping of ILCPs by using PLZF GFPcre/+ Rosa26-YFP reporter (PLZF-fm) mice (Constantinides et al., 2014). In these mice, ILCP progenies including ILC1s, ILC2s, and ILC3s rather than LTi or NK cells are preferentially labeled by YFP, though a certain ratio of blood cells expresses YFP due to the pre-hematopoietic PLZF expression, as reported previously (Constantinides et al., 2014). To remove this background YFP labelling, we sorted YFP − Lin − Sca1 + c-Kit + (YFP − LSK) cells from BM of PLZF-fm mice and transferred them into irradiated WT hosts. In these chimeric mice, AL 7 R − and 7R + ILC1s as well as other adult tissue ILC1s prominently expressed YFP ( Figure 1H and I). A similar trend was observed when using straight PLZF-fm mice or chimeric mice reconstituted with FL YFP − LSK cells from PLZF-fm mice (Figure 1-figure supplement 1H and I). Furthermore, FL G1-ILCs mostly expressed YFP in straight PLZF-fm mice ( Figure 1J), in line with a previous fate-mapping study of neonatal liver G1-ILCs (Constantinides et al., 2015). Thus, these results indicate that bona fide ILC1s lacking IL-7R are enriched in FL and AL.

7R + ILC1s minimally contribute to the development of 7R − ILC1s
A previous study reported that 7R + ILC1s behaved as the precursors of 7 R − ILC1s when cultured in vitro or transferred into lymphopenic mice (Friedrich et al., 2021). However, in the liver, while 7R + ILC1s were nearly absent in infants and accumulated with age, 7 R − ILC1s were predominant in young mice, decreased with age, and eventually depleted (Figure 2A-C). These observations suggest that To test this hypothesis, we first explored whether there were molecular pathways controlling the development of each ILC1 population individually. RNA-seq revealed that 7R + ILC1s highly expressed RORα and were positively enriched with gene sets 'RORA activates gene expression' relative to 7 R − ILC1s, based on gene set enrichment analysis (GSEA) ( Figure 2D). We therefore generated Rora -/mice to test the effect of RORα for 7R + ILC1s. As Rora -/mice tend to die within 4 weeks after birth, we analyzed adult Rora +/mice or 2 weeks old Rora -/mice. 7R + ILC1s were significantly reduced in Rora +/mice, while NK cells and 7 R − ILC1s were unchanged ( Figure 2E). In Rora -/mice, although whole ILC1s were significantly reduced in line with a recent study using Ncr1 Cre and Vav1 Cre Rora fl/fl mice (Song et al., 2021), 7R + ILC1s were the subset most apparently affected ( Figure 2F). These data suggest that the development of 7 R − ILC1s do not significantly depend on the presence of 7R + ILC1s.
To make the developmental relationships between 7 R − and 7R + ILC1s clearer, we conducted adoptive transfer experiments under physiological conditions by using unirradiated CD45.1 WT host mice. 7 R − and 7R + ILC1s were isolated from AL, transferred, and the host liver were analyzed. For at least 2 months, little conversion was observed between 7 R − and 7R + ILC1s ( Figure 2G-I), as evidenced by IL-7R and IL-18R1 expression ( Figure 2-figure supplement 1A). In addition, transferred BM iILC1s gave rise to AL 7R + ILC1s but not to 7 R − ILC1s (Figure 2-figure supplement 1B and C), consistent with their transcriptional resemblance. However, parabiosis experiments showed that the replacement rate of AL 7R + ILC1s were low (<5%), though significantly higher than that of 7 R − ILC1s (Figure 2figure supplement 1D), suggesting that both ILC1 subsets are tissue-resident. Thus, whether BM iILC1s actually contribute to AL 7R + ILC1 pool is still unclear. To test the phenotypical stability of ILC1s in inflammatory states, we injected IL-15/IL-15Rα complex repeatedly into host mice that had received Cell Proliferation Dye (CPD) eFluor 450-labeled 7 R − and 7R + ILC1s. NK cells and 7R + ILC1s proliferated more than 7 R − ILC1s after the stimulation ( Figure 2J), consistent with their basal Ki-67 expression levels ( Figure 1-figure supplement 1G) and properties of cytokine responsiveness (Figure 1-figure  supplement 1E). 7 R − and 7R + ILC1s were stable even after the IL-15/IL-15Rα stimulation ( Figure 2K), confirming their stability in activated states. These results show that 7R + ILC1s were rarely converted to 7 R − ILC1s and not essential for the development of 7 R − ILC1s under physiological conditions. FL G1-ILCs arise at hepatic parenchyma and give rise to 7R − ILC1s in sinusoids We next address the contribution of FL G1-ILCs to the development of AL 7 R − ILC1s. Adoptively transferred FL G1-ILCs differentiated into CD49a + CD49b lo mature ILC1s in AL ( Figure 3A) and they completely lacked IL-7R ( Figure 3B). To confirm the direct contribution of FL G1-ILCs to the adult pool of 7 R − ILC1s, we performed fate-mapping experiments using Ncr1-CreERT2 Tg mice (Nabekura and Lanier, 2016) crossed with Rosa26-tdTomato mice. After tamoxifen injection into pregnant mice and BM. Data represent three independent experiments (FL, n=15; AL, n=8; BM, n=6). (D) The percentages of IL-7R − fractions in non-NK G1-ILCs in the indicated tissues. LPL, small intestinal lamina propria lymphocytes; PC, peritoneal cavity; mLN, mesenteric lymph node; SG, salivary gland. Data are pooled from three independent experiments (FL,n=15;AL,n=8;BM,n=6;LPL,n=6;PC,n=6;SP,n=6;mLN,n=6;SG,n=6). (E) Scatter plot showing relative gene expression of FL G1-ILCs and AL 7 R − ILC1s compared to AL NK cells in RNA-seq. Genes differentially expressed by FL G1-ILCs, AL 7 R − ILC1s, AL 7R + ILC1s, and BM iILC1s (orange) or only by FL G1-ILCs and AL 7 R − ILC1s (blue) compared to AL NK cells are highlighted. FC, fold change. (F) First three principal components in PCA of top 3,000 variant genes. (G) Expression of T-bet (upper) and Eomes (lower) on FL G1-ILCs as well as NK cells, 7 R − ILC1s, and 7R + ILC1s in AL. Shaded histograms (grey) indicate isotype controls. Data represent two independent experiments. (H and I) Fate-mapping analysis of adult chimeric mice reconstituted with BM YFP − Lin − Sca1 + c-Kit + (LSK) cells from PLZF GFPcre/+ Rosa26-YFP (PLZF-fm) mice. Representative histograms of YFP expression (H) and the percentages of YFP + cells in indicated cell populations (I) are shown. Data represent or are pooled from two independent experiments (n=6 for AL T, AL NK, AL 7 R − ILC1, AL 7R + ILC1, and SP ILC1; n=3 for BM NK and BM iILC1). (J) The percentage of YFP + cells in FL G1-ILCs in E18.5 straight PLZF-fm mice. Data are from one experiment (n=5). RNA-seq data are from two (AL NK cells and SG G1-ILCs), three (AL 7R + ILC1s, BM iILC1s, and BM NK cells), and four (FL G1-ILCs, AL 7 R − ILC1s, and LPL ILC1s) biological replicates (E and F). Data are presented as mean ± SD.
The online version of this article includes the following source data and figure supplement(s) for figure 1: Source data 1. Fetal and adult liver contain bona fide ILC1s lacking IL-7R.   (Chen et al., 2022;Sparano et al., 2022). As shown in these studies, fate-mapped 7 R − ILC1s showed a skewed expression of Ly49E/F, though they also contained Ly49E/F − population (20-25%) (Figure 3-figure supplement 1B-1D). Labeling efficiency was 40% in neonatal IL-7R − G1-ILCs and 20% in 7 R − ILC1s in 4 weeks old mice ( Figure 3F and G). These results confirm a direct, albeit partial, contribution of FL G1-ILCs to the adult pool of 7 R − ILC1s.
To investigate the detailed developmental process of FL G1-ILCs and AL ILC1s in vivo, we examined their spatiotemporal distributions. In immunostaining analysis, FL G1-ILCs were identified as NKp46 + cells in WT mice ( Figure 4A). AL NK cells and ILC1s were identified as NKp46 + GFP − and NKp46 + GFP + cells in Cxcr6 GFP/+ mice, respectively ( Figure 4B and Figure 5-figure supplement 1A). In E18.5 liver, FL G1-ILCs mostly distributed at perivascular sites, outside of the sinusoidal lumen (here termed parenchyma; Figure 4A). In contrast, over 85% of whole ILC1s and NK cells were within sinusoids in AL ( Figure 4B and C). Although we could not detect IL-7R expression on ILC1s by immunofluorescence, flow cytometry (FCM)-based analysis of intravenous (i.v.) CD45.2 staining confirmed similar intravascular locations of 7 R − and 7R + ILC1s as well as NK cells, T cells, and NKT cells in AL ( Figure 4D and E). In contrast, ILC2s were not efficiently labeled by i.v. staining, consistent with perivascular localization of liver ILC2s observed so far (Dahlgren et al., 2019). Interestingly, a population of AL Lin -Sca-1 + Mac-1 + (LSM) cells, local precursors for ILC1s (Bai et al., 2021), were also not well labeled by i.v. staining. Thus, there are localization shifts between ILC1 precursors and mature ILC1s in the liver: FL G1-ILCs and some LSM cells distribute to parenchyma, whereas AL G1-ILCs including 7 R − ILC1s reside within sinusoids. These observations suggest that FL G1-ILCs arise at parenchyma and then infiltrate into sinusoids during maturation toward 7 R − ILC1s.
The online version of this article includes the following source data and figure supplement(s) for figure 2: Source data 1. 7R + ILC1s are dispensable for the development of 7 R − ILC1s in AL.  for G1-ILCs, we generated Lyve1 Cre/+ Il15 fl/fl mice, which target vascular IL-15 sources including sinusoidal endothelial cells and a fraction of hematopoietic cells (Lim et al., 2018;Pham et al., 2010). In Lyve1 Cre/+ Il15 fl/fl mice, all AL G1-ILC subsets were significantly reduced ( Figure 5F). We analyzed another mouse line targeting intravascular IL-15 sources, Lyz2-Cre Il15 fl/fl mice, which lack IL-15 in myeloid cells. Lyz2-Cre Il15 fl/fl mice showed similar two-fold reductions of AL NK cells, 7 R − ILC1s, and 7R + ILC1s ( Figure 5G), confirming the similar IL-15 requirements among all G1-ILC subsets. Notably, expression of Bcl-2, a survival factor downstream of IL-15, was downregulated in all G1-ILCs     Figure 5H and I), whereas Bcl-2 and Ki-67 levels were unchanged in Alb-Cre Il15 fl/fl mice ( Figure 5J and K). These results indicate that parenchymal IL-15 has no direct impact on mature 7 R − ILC1s in sinusoids whereas intravascular IL-15 directly supports the survival of all AL G1-ILCs. Alb-Cre Il15 fl/fl mice had reduced Lin − CD122 + CD49a + ILC1 precursors in AL ( Figure 5-figure  supplement 1D), suggesting an impaired development of 7 R − ILC1s. Thus, these data demonstrate that hepatocyte-derived IL-15 supports the development of 7 R − ILC1s at parenchyma, thereby maintaining AL 7 R − ILC1s infiltrated in sinusoids.

Steady-state mTOR activity confers granzyme B-mediated cytotoxicity in 7R − ILC1s
Cytotoxicity is one of the most pivotal functions of G1-ILCs, though the contribution of ILC1s remains controversial. By focusing on the ILC1 heterogeneity, we attempted to describe the G1-ILC effector function in detail. In steady state, minimal levels of granzyme B and death ligands were found on NK cells, while 7 R − ILC1s expressed both granzyme B and TRAIL, the latter of which was also expressed on 7R + ILC1s ( Figure 6A and B, Figure 6-figure supplement 1A, B; Friedrich et al., 2021). In line with this, freshly isolated 7 R − ILC1s remarkably lysed multiple tumor cells including YAC-1 ( Figure 6C), Hepa1-6 ( Figure 6D), and B16F10 cells ( Figure 6E). By contrast, NK cells and 7R + ILC1s showed only slight or no cytotoxicity against these tumor cells, consistent with a previous study showing minimal cytotoxicity of unstimulated NK cells (Fehniger et al., 2007). To determine the effector pathways 7 R − ILC1s rely on, we added concanamycin A (CMA), an inhibitor for perforin/granzyme pathways (Kataoka et al., 1994), and neutralizing antibodies for TRAIL and FasL to the coculture systems. Killing of Hepa1-6 cells by 7 R − ILC1s was markedly inhibited by CMA, and to a lesser extent by anti-TRAIL antibody ( Figure 6F). Despite the expression of granzyme A in NK cells and granzyme C in 7R + ILC1s Figure 6-figure supplement 1C and D), CMA had no effect to their cytotoxicity. Other granzyme genes (Gzmf, k, n, and m) were undetectable in ILC1s (data not shown). These results suggest that granzyme B plays a major role in the cytotoxicity of 7 R − ILC1s.
Cellular amount of granzyme B is well correlated to and primarily responsible for the NK cell cytotoxicity (Bhat et al., 2007;Gwalani and Orange, 2018;Prager et al., 2019). Although granzyme B expression and killing capacity of NK cells are weak at steady state, stimulation by cytokines, especially by IL-15, enable to induce both of them (Fehniger et al., 2007;Marçais et al., 2014;Prager et al., 2019). To test whether 7 R − ILC1s share such an activation machinery, we analyzed their expression of effector molecules after the stimulation. Expression levels of granzyme B, as well as TRAIL and granzyme C, in each G1-ILC subset were clearly upregulated by in vitro stimulation of IL-15 ( Figure 6-figure supplement 1E) and in vivo injection of IL-15/IL-15Rα complex ( Figure 6A and B, Figure 6-figure supplement 1A, and 1B). Interestingly, however, both IL-15 stimulation and IL-15/IL-15Rα injection enhanced granzyme B more efficiently on NK cells than 7 R − ILC1s, and thereby the granzyme B levels of NK cells overwhelmed or got comparable to those of 7 R − ILC1s ( Figure 6A and Figure 6-figure supplement 1E). IL-15/IL-15Rα injection also triggered the phosphorylation of STAT5, Akt, and ribosomal protein S6 (a target of mTOR), which are critical for the IL-15-induced effector function (Ali et al., 2015), in NK cells and 7R + ILC1s but to a lesser degree in 7 R − ILC1s ( Figure 6G and H, Figure 6-figure supplement 1F, and 1 G). These data suggest that the cytotoxic capacity of 7 R − ILC1s are different from that of NK cells in terms of responsiveness and requirement for the cytokine stimulation. Interestingly, the phosphorylation level of S6 was rather higher in ILC1s than NK cells in unstimulated mice ( Figure 6G and H). Notably, injection of rapamycin, an mTOR complex inhibitor, downregulated granzyme B expression in 7 R − ILC1s to a level comparable to that in NK cells ( Figure 6I and J). By contrast, granzyme B levels in NK cells and 7R + ILC1s were unaffected. Collectively, these results show that 7 R − ILC1s exhibit cytotoxicity in their steady state through granzyme B expression, which is supported by their constitutive mTOR activation.
The online version of this article includes the following source data for figure 4: Source data 1. Liver G1-ILCs shift distributions from parenchyma to sinusoids during development.

Discussion
In this study, we have characterized the developmental process and functional heterogeneity of liver G1-ILCs. Hepatocytes shape IL-15 niches supporting parenchymal development of FL G1-ILCs, that differentiate into 7 R − ILC1s in sinusoids. Functionally, 7 R − ILC1s exhibit granzyme B-mediated cytotoxicity in steady state, in sharp contrast to less cytotoxic resting NK cells. ILC1 heterogeneity has been extensively addressed recently. In the liver, ILC1s are separated into IL-7R − and IL-7R + populations (Friedrich et al., 2021;Sparano et al., 2022;Yomogida et al., 2021), the latter of which can differentiate into the former when cultured in vitro or transferred into lymphopenic mice (Friedrich et al., 2021). However, we show that 7 R − and 7R + ILC1s behave like independent subsets under physiological conditions: decline of 7 R − ILC1s and accumulation of 7R + ILC1s with age, requirements for RORα specifically in 7R + ILC1s, and phenotypical stability between 7 R − and 7R + ILC1s when transferred into WT host mice. Such a contradiction might be due to the highly nutrient-and cytokine-accessible environments in the culture systems and lymphopenic hosts that might trigger non-physiological activation and phenotypic shift of 7R + ILC1s. Indeed, our model rather gives an explanation for the ILC1 heterogeneity in inflammatory disease models using healthy mice observed so far. In mouse models of contact hypersensitivity, MCMV infection, and liver injury, ILC1s with high expression of cytokine receptors (IL-7R, CD25, and/or IL-18R) highly proliferate and accumulate in AL, thereby forming the memory and protecting liver from infection and injury (Nabekura et al., 2020;Wang et al., 2018;Weizman et al., 2019). AL 7R + ILC1s resemble such 'memory-like' or 'activated' ILC1s in terms of the surface phenotype and high proliferation potentials. These observations suggest a hypothesis that a preferential proliferation of pre-existing stable 7R + ILC1s, rather than inflammation-specific ILC1s induced from naïve ILC1s, may contribute to liver immunity and homeostasis.
Several previous studies have pointed out the precursors for ILC1s: BM iILC1s (Klose et al., 2014), FL G1-ILCs (Constantinides et al., 2015;Daussy et al., 2014), and local precursors in the liver such as LSM cells and Lin − CD122 + CD49a + cells (Bai et al., 2021). In particular, FL G1-ILCs are precursors for AL Ly-49E + ILC1s (Chen et al., 2022;Sparano et al., 2022). Although AL Ly-49E + ILC1s are included in and account for 30-40% of AL 7 R − ILC1s, fate-mapping reveals that FL-derived 7 R − ILC1s contain also an Ly49E/F − population (20-25%), suggesting further heterogeneity in FL-derived ILC1s. Considering the partial contribution (about 50%) of FL G1-ILCs to AL 7 R − ILC1 pool estimated by fate-mapping, local ILC1 precursors such as LSM cells and Lin − CD122 + CD49a + cells might be the other sources for 7 R − ILC1s. By contrast, the origin of AL 7R + ILC1s remains to be solved. We show that BM iILC1s have a potential to differentiate into AL 7R + ILC1s, but the actual contribution is unclear. As both AL 7 R − and 7R + ILC1s were rarely replaced during parabiosis experiments using adult mice, it is possible that a transiently migrated population derived from BM settle and give rise to 7R + ILC1s in the liver during neonatal period, as discussed previously (Sparano et al., 2022). Further investigations using a specific tracing approach such as fate-mapping of BM iILC1s are required to determine their precise developmental potency.
The online version of this article includes the following source data and figure supplement(s) for figure 5: Source data 1. Hepatocytes provide the parenchymal IL-15 niche regulating the local development of 7 R − ILC1s. The percentages of annexin Ⅴ + PI + YAC-1 cells in flow-based cytotoxicity assays. Freshly isolated effector cells were co-cultured with target cells for 4 hours (E:T ratio = 10:1). Data are pooled from two independent experiments (NK, n=7; 7 R − ILC1, n=10; 7R + ILC1, n=6). (D and E) Target cell viability at each timepoint in time-lapse cytotoxicity assay using Hepa1-6 cells (D) (two independent experiments; n=6 for NK, n=6 for 7 R − ILC1, and n=4 for 7R + ILC1 in all timepoints) and B16F10 cells (E) (two independent experiments; n=7 for NK, n=8 for 7 R − ILC1, and n=8 for 7R + ILC1 in all timepoints) as target cells. Freshly isolated effector cells were co-cultured with target cells up to 6 hours (E:T ratio = 10:1). (F) Hepa1-6 cell viability at 320 min in time-lapse cytotoxicity assays supplemented with concanamycin A (CMA) or neutralizing antibody for TRAIL (α-TRAIL) or FasL (α-FasL) compared to vehicle-supplemented controls. Data are pooled from two independent experiments (NK, n=6; 7 R − ILC1, n=6; Figure 6 continued on next page partly due to the immaturity of hepatic vasculature in that period. Since FL G1-ILCs and FL HSCs are also similar in that they eventually infiltrate into blood vessels (Lewis et al., 2021), it would be of interest to address the mechanism underlying their neonatal dynamics. In addition, it is still unclear why hepatocyte-derived IL-15 has such a local effect despite many fenestrae and the lack of a basement membrane on liver sinusoids. One possible explanation is that the transpresentation of IL-15/ IL-15Rα by hepatocytes may require direct contact to target cells, as dendritic cells do (Mortier et al., 2008). Given that hepatocytes prominently express Il15ra gene and its deletion results in the reduction of whole IL-15-dependent lymphocytes in the liver (Cepero-Donates et al., 2016), it is also possible that hepatocytes may produce IL-15Rα as a soluble form, that binds to other cell-derived IL-15 to exert non-local effects.

Figure 5 continued
Traditionally, ILC1s are regarded as less cytotoxic than NK cells in mice, whereas recent studies have challenged this theory (Dadi et al., 2016;Di Censo et al., 2021;Kansler et al., 2022;Nixon et al., 2022;Yomogida et al., 2021). Our study provides two possible explanations for this discrepancy. First, the age of mice selected for analysis influence the composition and overall cytotoxicity of ILC1s. We and others (Chen et al., 2022;Friedrich et al., 2021;Nixon et al., 2022) showed that ILC1s were heterogenous in their cytotoxicity. Due to the age-dependent reduction of highly cytotoxic 7 R − ILC1s, the overall cytotoxicity of ILC1s in the liver should decline with age. Second, the effector program of 7 R − ILC1s differ from NK cells in its nature, especially in terms of cytokine responsiveness. Freshly isolated NK cells exhibit low expression of cytotoxic molecules and only minimal cytotoxicity (Fehniger et al., 2007), while stimulation by IL-15 confers granzyme B expression and cytotoxicity on NK cells via mTOR-dependent metabolic reprogramming (Marçais et al., 2014;Nandagopal et al., 2014). Conversely, we show that 7 R − ILC1s exhibit prominent granzyme B-mediated cytotoxicity via mTOR activity at steady state, though they are less responsive to cytokines than NK cells and 7R + ILC1s. These findings suggest that 7 R − ILC1s are 'ready-to-kill' sentinels that contribute to the tonic immune surveillance, which is followed later by the response of activated and proliferated NK cells and 7R + ILC1s. While the meaning of the predominance of such a cytotoxic subset especially in early life is unknown, it is possible that FL G1-ILCs and 7 R − ILC1s play some physiological roles in the early liver development or maturation by eliminating unnecessary cells.
Taken together, our study provides insight into the complex ILC1 ontogeny by revealing relationships among heterogenous ILC1 subsets, their developmental dynamics, and niche dependence. Our findings highlight the intrinsic cytotoxic programs of 7 R − ILC1s unlike NK cells, proposing them as critical steady-state sentinels against infection prevention and tumor surveillance and bringing the possibility of local therapeutic targeting of ILC1 function.
The online version of this article includes the following source data and figure supplement(s) for figure 6: Source data 1. 7 R − ILC1s exhibit cytotoxicity via granzyme B expression underpinned by steady-state mTOR activation.   (Pham et al., 2010) kindly supplied by Dr. Jason Cyster at University of California San Francisco, and Lyz2-Cre Tg mice were bred with Il15 flox/flox (Il15 fl/fl ) mice, which were generated in our laboratory (Cui et al., under review). Ncr1-CreERT2 Tg mice (Nabekura and Lanier, 2016) were provided by Dr. T. Nabekura and Dr. Lewis L. Lanier and crossed with Rosa26-tdTomato (Madisen et al., 2010) mice. For fetal experiments, the noon when the vaginal plug was detected was considered as embryonic day (E) 0.5. All mice were maintained under specific pathogen-free conditions in the Experimental Research Center for Infectious Diseases at the Institute for Life and Medical Sciences, Kyoto University. All procedures were carried out under sevoflurane or isoflurane anesthesia to minimize animal suffering. All mouse protocols were approved by the Animal Experimentation Committee of the Institute for Life and Medical Sciences, Kyoto University.

Cell preparation and isolation
To protect ILC1s from NAD + -induced cell death (NICD) (Stark et al., 2018), mice were intravenously (i.v.) injected with 40 μg ARTC2.2 blocking nanobody (BioLegend, San Diego, CA, USA) 30 min before sacrificing the mice in several experiments. Fetal liver, adult liver, spleen, peripheral (axillary, brachial, and inguinal) lymph nodes, and mesenteric lymph nodes were dissociated mechanically and passed through 70 μm cell strainers (Greiner Bio-One, Milan, Italy). Adult liver leukocytes were then separated by centrifugation through 40% Percoll. Peritoneal cavity was washed by 5 mL of PBS and the wash fluid was extracted using a syringe and a 21 G needle (Terumo Corporation, Tokyo, Japan). BM cells were obtained by flushing out the marrow fraction of femurs and tibias. To collect salivary gland cells, submandibular and sublingual glands were minced with scissors and incubated at 37℃ for 1 hr in RPMI 1640 medium containing 10% fetal bovine serum, 1 mg/mL collagenase D, and 50 μg/mL DNase I (Sigma-Aldrich, St. Luis, MO, USA). The cell suspension was filtered through a 70 μm cell strainer and purified using 40% Percoll. For the isolation of intestinal lamina propria lymphocytes, small intestines were flushed out and Peyer's patches were excised. The intestines were opened longitudinally, cut into 1 cm pieces, and incubated at 37℃ for 30 min in PBS with 5 mM EDTA to remove epithelial cells. The incubated pieces were then minced and digested by RPMI 1640 medium containing 10% fetal bovine serum, 1.25 mg/mL collagenase D, and 50 μg/mL DNase I. The tissue suspension was passed through a 70 μm cell strainer and lymphocytes were purified by 40% Percoll. . For intracellular staining of Eomes, T-bet, Bcl-2, Ki-67, and granzymes, cells were stained for surface antigens, fixed, permeabilized, and stained using Foxp3 Staining Buffer Set or IC Fixation Buffer (Thermo Fisher Scientific). For intracellular staining of p-S6, p-STAT5, and p-Akt (S473), cells were stained for surface antigens, fixed, permeabilized, and stained using BD Phosflow Buffer (BD Biosciences). Flow cytometry and cell sorting were performed on BD FACSVerse or BD LSRFortessa X-20 flow cytometers (BD Biosciences) and BD FACS Aria Ⅱ or Aria Ⅲ cell sorters (BD Biosciences), respectively. Data were analyzed on FlowJo software (FlowJo, Ashland, OR, USA). Debris and dead cells were excluded from analysis by forward and side scatter and propidium iodide (PI) gating. In figures, values in quadrants, gated areas, and interval gates indicate percentages in each population.

RNA sequencing (RNA-seq) and data analysis
For bulk RNA-seq, freshly sorted G1-ILC populations (1×10 3 cells) were lysed with Buffer RLT (Qiagen, Hilden, Germany) and purified with RNAClean XP (Beckman Coulter, Brea, CA, USA). Double strand cDNA was synthesized, and sequencing libraries were constructed using SMART-seq HT Plus kit (Takara Bio, Otsu, Japan). Sequencing was performed with 150 bp paired-end reads on the Illumina HiSeq X sequencer (Illumina, San Diego, CA, USA). fastp (Chen et al., 2018) was used to assess sequencing quality and to exclude low-quality reads and adaptor contaminations. Reads were mapped on the mouse reference genome (mm10) using HiSat2. The read counts were determined at the gene level with featureCounts. Normalization of gene expression levels and differential gene expression analysis were performed using DESeq2. Genes were considered as differentially expressed genes (DEG) when they had an adjusted p (p adj ) value <0.05 and fold changes >1.0. Metascape (Zhou et al., 2019) and gene set enrichment analysis (GSEA, Broad Institute) was used for enrichment analysis. For reanalysis of single nuclei RNA-seq (snRNA-seq) data of whole liver cells in mice (Liver Cell Atlas; https://www. livercellatlas.org/), normalization, scaling, and UMAP clustering using first 5 dimensions in principal component analysis (PCA) of scaled count matrix were performed on R package Seurat 4.0.2.

In vivo treatment
For in vivo stimulation of G1-ILCs, mice were administrated intraperitoneally (i.p.) with 2 μg IL-15/ IL-15Rα complex (the RLI form as in Mortier et al., 2008, provided by Dr. J. M. Dijkstra) once a day. After 18 hr, liver cells were isolated and analyzed by flow cytometry to detect cytotoxic molecule expression. For rapamycin treatment, 30 μg rapamycin in 100 μL corn oil was injected i.p. into mice 18 hr before the analysis.

Intrasplenic injection
A small incision was made on the left flank of anesthetized mice and the lower pole of the spleen was gently exposed. Cell suspension (50 μL) was slowly injected into the spleen by a 0.3 mL insulin syringe with a 29 G needle (BD Biosciences). Cotton wool was applied to the spleen for several minutes after the injection to stop bleeding.

Parabiosis
Female CD45.1 and CD45.2 congenic C57BL/6 J mice were surgically conjoined as previously described (Gasteiger et al., 2015). In brief, lateral skin from elbow to knee of each mouse was sutured, forelimbs and hindlimbs were tied together, and the skin incisions were closed using surgical adhesive. After 60 days of surgery, mice were analyzed by flow cytometry.

Cell lines
Hepa1-6 cells were purchased from RIKEN BioResource Center (RIKEN BRC, Tsukuba, Japan), which is authenticated by STR profiling (https://www.cellosaurus.org/CVCL_0327). B16F10 cells were provided by Dr. T. Honjo at Kyoto University. Both cell lines were confirmed to be mycoplasma negative before use, and were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 2 mM L-glutamine, and antibiotics.

In vitro killing assay
For time-lapse killing assays, 2×10 2 Hepa1-6 cells or B16F10 cells were labeled by CPD eFluor 450 and pre-incubated with RPMI 1640 medium (without phenol red) containing 10% FBS, 10 mM HEPES (pH7.4), antibiotics, and 1 μg/mL PI on 96-well round bottom plates. Freshly sorted liver NK cells, 7 R − ILC1s, or 7R + ILC1s (2×10 3 cells) were added to the plates and co-cultured with tumor cells. Timelapse imaging was performed using a BZ-X710 microscope (Keyence, Osaka, Japan) with CFI Plan Apo λ 10×and CFI Plan Fluor DL 10×objective lenses at a 20 min interval for up to 6 hr. To determine the contribution of effector molecules, 50 nM concanamycin A (CMA), 10 μg/mL anti-TRAIL antibody (N2B2), or 10 μg/mL anti-FasL antibody (MFL3) were supplemented and compared to vehiclesupplemented controls. Tumor cell viability was defined by the ratio of the CPD + PI − viable tumor cell number at each time point to the viable tumor cell number at the beginning of the imaging and represented as the moving average of three consecutive time points. Image analysis and cell counts were performed using BZ-X Analyzer (Keyence). For killing assay of YAC-1 cells (provided by Dr. M. Hattori at Kyoto University), 2×10 4 freshly sorted liver NK cells, 7 R − ILC1s, or 7R + ILC1s were co-cultured for 4 hr with 2×10 3 CPD eFluor 450-labeled YAC-1 cells in RPMI 1640 medium containing 10% FBS, 10 mM HEPES (pH7.4), and antibiotics. After culture, YAC-1 cells were stained with FITC-conjugated Annexin V and PI (MEBCYTO-Apoptosis Kit, MBL) and the ratio of Annexin V + PI + apoptotic cells were analyzed by flow cytometry.